MICR3051finalexamstudyguide.pdf MICR 3050
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This 9 page Study Guide was uploaded by jj on Thursday September 3, 2015. The Study Guide belongs to MICR 3050 at Clemson University taught by Kristi James Whitehead in Spring 2015. Since its upload, it has received 213 views. For similar materials see General Microbiology in Biological Sciences at Clemson University.
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Date Created: 09/03/15
Haley Poole MICR 3051 Final Laboratory Exam Study Guide 1 Describe how UV light is germicidal its effect on DNA and the wavelength at which UV light is most lethal In general for electromagnetic radiation the shorter the wavelength the more damaging it is to cells thus UV light is much more germicidal than either visible light or infrared radiation Most bacteria are killed by the effects of ultraviolet light and UV light is routinely used to sterilize surfaces The primary lethal effects of ultraviolet light are due to its mutagenic properties UV radiation at 260nm is the most germicidal because its wavelength is the specific wavelength at which DNA maximally absorbs UV light When DNA absorbs UV light it causes the formation of pyrimidine dimers These form when a covalent bond is formed between two adjacent thymine or cytosine molecules in a DNA strand Dimers essentially cause DNA molecule to become deformed so that the DNA polymerase cannot replicate DNA strands past the site of dimer formation nor can genes past this point be transcribed 2 Describe how we examined the germicidal effects of UV on S aureus and B subtilis S aureus is a nonendospore former and B subtilis is an endospore former One half of each plate will be shielded from the radiation to provide a control comparison B subtilis and S aureus will be used to provide comparison of the relative resistance of vegetative cells and endospores We swabbed the bacteria all over the nutrient agar Then covered half of each plates with a paper towel and placed them under the UV light After exposing the plate for our assigned exposure times we put the lid back on the plate and placed it in the incubator inverted for 2448 hours at 37 C Then the growth of the exposed side of the plate was observed and recorded in the results as substantial growth moderate growth three or fewer colonies or no growth 3 Be able to define the following terms antiseptic disinfectant steriliant sanitizer bacteriostatic and bacteriocidal Antiseptic substances that inhibit microbial growth or kill microorganisms Gentle enough to be applied to living tissue Do not destroy endospores Some antiseptics are classified as drugs and are therefore regulated by the Food and Drug Administration Disinfectant chemical agents that are applied to inanimate objects to kill microorganisms More harsh than antiseptics and are damaging to living tissues Steriliant destroy all microbial life including endospores Sanitizer agents that reduce microbial numbers to a safe level but do not completely eliminate all microbes Bacteriostatic agents that inhibits the growth of bacterial cells but doesn t kill them Bacteriocidal agents that kill bacterial cells Can be specific in terms of groups of microorganism there are bactericides fungicides and viricides 4 Explain the filter Paper Disk Method of evaluating the relative effectiveness of various disinfectants andor antiseptics In this procedure a plate of suitable medium is streaked with the test organism Filter paper disks the same size as antibiotic disks are impregnated with the agent by dipping the disk in a germicide or disinfectant and placing the disk on an inoculated plate The plate is incubated for 48 hours The agent will diffuse from the disk into the agar forming a concentration gradient If the substance is inhibitory a clear zone of inhibition will surround the disk where no growth has occurred The size of the zone can be used to quantitatively compare one agent s effectiveness against other chemical substances 5 Distinguish between antibiotic and antimicrobials Antimicrobials are compounds that kill or inhibit microorganisms Antibiotics are antimicrobials usually of lower molecular weight produced by microorganism that inhibit or kill other microorganisms 6 Explain the KirbyBauer method of evaluation the sensitivity of bacteria to certain antibiotics and or antimicrobials KirbyB auer method is used to determine the sensitivity or resistance of a bacterium to an antimicrobial This is a standardized test procedure that is reliable relatively simple and yields results in a short time It is performed by uniformly streaking a standardized inoculum of test organism on Mueller Hinton medium and then paper disks containing specific concentrations of an antimicrobial or antibiotic are deposited on the agar surface The antimicrobial diffuses out from the disk into the agar forming a concentration gradient IF the agent inhibits or kills the test organism there will be a zone of inhibition The zone can vary with diffusibility of the agent the size of the inoculum the type of medium and other factors High potency disks are used that may be placed on the agar with sterile forceps To secure the disks to the medium it is necessary to press them down into the agar After 1618 hours of incubation the plates are examined and the diameters of the zones of inhibition are measured to the nearest millimeter Diameters for tested bacteria are compared to those in a table that are based on values obtained for ATCC American Type Culture Collection reference cultures that are sensitive to the specific antibiotic Cultures are designated as resistant sensitive or intermediate in their responses to the antibiotic 7 Understand how you would measure a zone of inhibition what a zone of inhibition is and how you would determine if a bacterium is R I or S to an antimicrobial agent Zone of Inhibition the zone around the disk where no growth occurs You measure a zone of inhibition with a metric ruler to the nearest whole millimeter The zone of complete inhibition is determined without magnification To determine which antibiotics your organism is sensitive to S or resistant to R or intermediate 1 consulate the table on page 151 of lab manual It is important to note that the significance of zone of inhibition varies with the type of organism 8 Describe the effects of alcohol on bacterial growth Alcohol kills bacteria 9 Explain the basis of the Reductase Test and how it can be used to distinguish between high and low quality milk Know the meaning and significance of MBRT Milk contains large numbers of actively growing bacteria will have a lowered oxidationreduction potential due to the exhaustion of dissolved oxygen by microorganisms This lowered oxidationreduction potential can be measured by the addition of the dye methylene blue to a milk sample which is the basis for the reductase test of milk The time it takes for the methylene blue to become colorless is the methylene blue reduction time MBRT The shorter the MBRT the lower the quality of milk An MBRT of 6 hours is very good whereas an MBRT of 30 minutes is of very poor quality 10 Understand the role that microbes have in food production and in food spoilage The presence of microorganisms in food does not necessarily indicate that the food is spoiled or that it has the potential to cause disease High bacterial counts in food do not necessarily mean that the food is spoiled or that it harbors diseasecausing organisms but it can suggest the potential for more rapid spoilage in food Pasteurization and smoking will significantly reduce the numbers of bacteria present in food Pasteurization kills many of the bacteria that are introduced during processing and any pathogens that may be present but it does not kill all the bacteria present in milk Some bacteria in milk can survive pasteurization temperatures and eventually cause spoilage and souring of milk 11 Understand how to conduct a Standard Plate Count including determining in a dilution scheme to determine bacterial numbers in a liquid or solid food sample Standard plate count SPC or viable count is one of the most common methods for determining bacterial numbers in a sample A sample is diluted in a series of dilution blanks Aliquots of dilutions are then placed onto media and the numbers of colonies are counted after incubation of 2448 hours It is assumed that the bacterial cells are diluted to an end point where a single cell divides giving rise to a visible colony on a plate The number of bacteria in the original sample is determined by multiplying the number of colonies by the dilution factor SPC are reported as colony forming units CFUs Only numbers between 30 and 300 CFUs are considered statistically valid 12 Be able to calculate the number of bacteria in a sample based on the number of CFUs on various plates Know what constitutes a countable plate 30 to 300 colony forming units constitutes as a countable plate CFUmL CFU x Dilution Factor Volume Culture Plated 13 Explain how the grape juice fermentation ask was set up and know what observations would indicate fermentation had occurred 100ml of grape juice is inoculated with 3ml of yeast culture pH of juiceyeast mixture is determined before incubation Lead acetate teststrip is taped to inside of ask neck The mouth of ask is sealed with rubber balloon before incubation The ask is incubated at room temperature for 7 days Balloon is removed after incubation Odor of gas and test strip change are noted pH of fermented juice is checked after incubation 14 Define the process of ammonification When living organisms die their remains are decomposed in the soil by a variety of microorganisms Proteins are degraded by proteases liberating amino acids in which the amino group is removed by deamination to release ammonia Nucleic acids are also degraded releasing ammonia and animal wastes such as uric acid are degraded to produce ammonia The process of ammonia production in the soil is called ammonification Bacteria and plants can assimilate the ammonia to synthesize new proteins and nucleic acids for growth 15 Describe how to test for the presence of ammonia in soil samples Inoculate one tube of peptone broth with a small amount of soil Incubate the tube at room temperature for 34 days After 3 to 4 days test the medium for ammonia Deposit a drop of Nessler s Reagent into two separate depressions of a spot plate Add a loopful of the inoculated peptone broth to one depression and a loopful from the sterile not inoculated tube in the other Interpretation of ammonia presence is as follows Faint yellow color small amount of ammonia Deep yellow more ammonia Brown precipitate large amount of ammonia 16 Describe coliforms and how they are good indicators of fecal contamination in water Coliforms are gramnegative facultative anaerobic nonendospore forming rods that ferment lactose to produce acid and gas in 48 hours at 35 C E coli is routinely found in human intestine but is not found in soil or water is a good indicator of fecal contamination in water because 1 it occurs primarily in the intestines of humans and warm blooded animals 2 the organism can be easily identified by microbial tests 3 it is not as fastidious as the intestinal pathogens and it survives a little longer in water samples 17 Explain how the presumptive and confirmed tests are conducted and how they are used to determine the presence of coliforms in water sample Know the media used for each and what a positive test looks like The presumptive test is 15 tubes of lactose broth that are inoculated with measured amounts of water to see if it has any lactose fermenting bacteria that produce gas If after incubation gas is seen it is presumed that coliforms are present in the This test is also used to determine the MPN of coliforms present per 100 ml of water Confirmed test plates of Levine EMB agar and Endo agar are inoculated from the positive tubes to see if the organisms that are producing gas are gram negative Both agars inhibit the growth of gram positive bacteria and cause colonies of coliforms to be distinguishable from noncoliforms EMB agar coliforms produce small colonies with dark centers Endo agar coliforms produce reddish colonies that are sometime metallic green 18 Know how to determine the MPN of water sample Thomas simple formula MPN100ml no positive tubes X 100 w ml sample in X ml sample in negative tubes all tubes 19 Describe the distinguishing characteristic of bacteriophages Bacteriophages are viruses that infect bacterial cells Like all viruses bacteriophages or phages for short are obligate intracellular parasites that is they must invade a host cell in order to replicate and reproduce This is due to the fact that viruses are composed primarily of only a single kind of nucleic acid molecule encased in protein coat or capsid that protects the nucleic acid A phage consists of a nucleocapsid the nucleic acid and protein capsid sheath protein that nucleocapsid is attached to base plate what the sheath sits on tail bers and spikes A single virus or phage particle is called a virion 20 Explain the procedure for determining a bacteriophage titer Recognition a bacteriophage recognizes its host by its tail fibers binding to chemical groups associated with receptors on the surface of the host cells These receptors perform other functions for the bacterial cell If the group that are recognized by tail fibers are not present on a bacterial cell a phage cannot bind to the cell and cause infection Penetration The phage particle settles onto the surface of the host cell and lysozyme that is associated with the phage tail begins to erode a localized area of the cell wall thus weakening it The sheath contracts forcing a hollow core through the weakened area of the cell wall As contraction occurs the viral nucleic acid is injected through the core into the bacterial cytoplasm Replication Release Two types Temperatelysogenic phage and lytic phage The host cell is not always going to be ruptured immediately sometime it can take a long time sometimes it will never rupture Options are lytic pathway and lysogenic pathway Depends on conditions around the cell health of the cell as to which pathway will happen Lytic pathway act as virulent phage Once lytic pathway is started virus will become active make host cell make large numbers of virus and the host cell will lyse in order to release viral particle Lysogenic pathway virus will stay in host cell without destroying it Most common way to do this is for lysogenic phage to incorporate its DNA into host cell DNA Virus will inject DNA into host cell cytoplasm viral DNA will get incorporated into host cell chromosome Benefit to virus as the host cell genome is getting replicated the virus genome is being replicated too No big problem with this pathway until there is a trigger that causes this virus to enter the lytic pathway I process of entering lytic pathway is called induction 21 Know how to calculate the number of phage particles in a suspension by counting the number of PFUs on plates PFUml Number of plaques counted Dilution X Volume of diluted virus added to the plate 22 Describe the two mutant isolation procedures we used and know what types of mutants we were trying to isolate Mutations occur in one of two ways 1 Spontaneous mutations arise occasionally in all bacteria and develop in the absence of any added agent 2 Induced mutations are the result of the bacterium s exposure to a mutagen which is a physical or chemical agent Spontaneous mutations resistant to antibiotics such as streptomycin are easily detected because they grow in the presence of the high antibiotic concentrations that inhibit the growth of normal bacteria this is indicative of streptomycin resistant mutant The gradient agar plate method is to isolate and select streptomycin resistant mutant E coli Mutation by replica plating organisms are spread over nutrient agar with a sterile bent glass rod After incubation colonies are picked up with velveteen colony carrier Nutrient agar is inoculated by lightly pressing carrier into it Streptomycin agar is inoculated with same carrier in same manner By comparing the two plates it can be determined which of the colonies on the original plate are spontaneous mutants to streptomycin 23 Know the purpose of the gradient plate and the three plates used in the replica plating Replica plating can determine which of the colonies on the original plate are spontaneous mutants to streptomycin from comparing the other two plates to the original Gradient plate observe plate for development of resistant bacteria colonies in the areas of higher streptomycin concentration 24 Describe the general characteristics of Staphylococcus aureus as part of the micro ora of the human body and its significance in disease Although staphylococci were originally isolated from pus in wounds they were later demonstrated to be part of the normal microbiota of nasal membranes hair follicles skin and the perineum in healthy individuals Staphylococci are initiated when a breach of skin or mucosa occurs when a host s ability to resist infection occurs or when a staphylococcal toxin is ingested The most notable virulence factor is coagulase production Virtually all strains of S aureus are coagulase positive and will cause serum to form a clot The role of coagulase in the pathogenies of disease is unclear but coagulase may cause a clot to form around the staphylococcal infection thus protecting it from host defenses S aureus also is associated with DNase a nuclease that digests DNA S aureus produces a hemolysin called alpha toxin that causes a wide clear zone of betahemolysis on blood agar This toxin plays a significant role in virulence because it lyses red blood cells and damages leukocytes heart muscles and renal tissue S aureus ferments mannitol to produce acid 25 Describe how we isolated S aureus from our noses and how we can identify it using blood agar MSA and CHROMagar Swabbed our noses using the sterile cotton swab Inoculated the first quadrant of the blood agar plate with the sample Using a sterile inoculated loop streak for isolation into the second third and fourth quadrant of the plate Repeat steps 13 using MSA plate Incubate the plates at 37 C for 48 hours observe and then refrigerate the plates until the next lab Evenly divide your CHROMagar plate by drawing a line down the middle of the bottom of the plate Pick an isolated betahemolyitc colony from your blood agar plate or an isolated colony from your MSA plate using a sterile inoculating loop Streak your colony on half of the CHROMagar plate Incubate plate at 37 C for 48 hours observed 26 Define transformation The third method of recombination it occurs when a bacterial cell receives small amounts of DNA from the environment Transformation can also be used in the biotechnology lab to add new genes to bacteria creating recombinant cell that then act as biological factories expressing any protein encoded by their newly acquired DNA Key to the transformation of cells in the lab is the use of small circular piece of DNA called plasmid Plasmid is typically a few thousand nucleotides in length and contains several areas of interests including origin of replication ori drug resistance gene of interest promoter 27 Know the four basic steps to isolate DNA from bacterial cells The bacterial cells are centrifuged to concentrate them A detergent is used to break down the cell wall and cell membrane releasing DNA A protease along with heat is used to denature and digest proteins within the cell Cold alcohol is used to precipitate the DNA from the solution allowing it to be spooled onto a glass or metal rod 28 Describe the heat shock method we used to artificially transform Ecoli with pGLO We marked the bottom of each petri dish with the media it contains LB LBamp or LBampara Label two microcentrifuge tubes with your group number and label one tube pGLO and pGLO Using a sterile transfer pipet add 250 111 transformation solution to each tube and place the tubes on ice Using a sterile loop transfer an entire colony of E coli to the pGLO tube Use the loop to completely disperse the cells in the transformation solution Place the tube back on ice Repeat this process for the pGLO tube closing the lid on the tube before returning it to the ice Use a supplied sterile loop to transfer a loopful of the pGLO DNA solution to the pGLO tube Mix the DNA solution with the cell suspension in the tube Close the lid of the tube and return it to the ice Dispose of the pipet Incubate the tubes on ice for 10 min If the tubes are in a tube holder be sure that they make good contact with the ice Transfer from tubes from the ice bath to a water bath set at 42 C Allow the tubes to remain in the water for exactly 50 seconds and return them to the ice For best results the temperature change from cold to hot and back to cold must be as rapid and complete as possible Continue incubating the tubes for the ice for a total of 2 minutes Using a sterile pipet add 250111 LB broth to the pGLO tube and close the top Repeat this process with a second pipet for the other tube Allow both tubes to incubate at room temperature for 10 minutes Mix the tubes gently by tapping with your finger Using a new sterile pipet for each transfer inoculate the plates pGLO 200111 onto LB plate pGLO 200111 onto LBamp plate pGLO 200111 onto LBamp plate pGLO 200111 onto LBampara plate Using a sterile loop to spread the transformation mixture over the entire surface of the plate Repeat this process for the other three plates using a sterile loop each time Incubate the plate at 37 C for 24 hours 29 Know the selectable characteristics of pGLO that allowed us to select for transformants pGLO contains ori the bla gene which provides resistance to ampicillin and the gene that codes for the production of a green uorescent protein GFP Transcription of the GFP gene is controlled by an arabinose promoter 30 Know the role of araC in pGlo and why arabinose was added to one of the plates When arabinose is present in the environment it binds to a DNAbinding protein called araC In the presence of arabinose araC allows the protein RNA polymerase to bind to the promoter and transcribe the genes which will in turn be translated to provide ampicillin resistance and green uorescent protein If no arabinose is present the genes will not be transcribed or translated 31 Describe the effects of lysozyme on bacterial cells Lysozyme degrade the B 14 bond between the amino sugar molecules in peptidoglycan thus causing breaks in the lattice and weakening the cell wall As a result the solute pressure of the cytoplasm can cause the cell membrane to rupture through these breaks resulting in cell lysis and death 32 Be able to define and calculate morbidity mortality and incidence Morbidity illness due to a specific disease Mortality is the second epidemiological measurement and refers to the number of deaths within a specified period among people having a particular disease Incidence compares the number of new cases of a disease during a specified period to the size of the susceptible population during that period Morbidity number of cases per period X K susceptible population size at midpoint of period Mortality Number diseaserelated deaths per period Number people with the disease X K Incidence number new cases susceptible population size X K at midpoint of period 33 Define communicable epidemiology endemic epidemic and pandemic Communicable infectious diseases that are transmissible to other persons Epidemiology the study of how when where what and who are involved in the spread and distribution of diseases in human populations Endemic an infectious disease that exhibits a steady frequency over a long period of time in a particular region Epidemic number of newly reported cases in a given period of time in a specific area is excessive Pandemic disease spreading to one or more continents 34 Define antigen antiserum serological typing and agglutination Antigen any substance that causes your immune system to produce antibodies against it Antiserum a serum containing antibodies Serological typing a technique based upon antibodyantigen reactions by which pathogenic bacteria are identified Agglutination occurs if an antigen is mixed with its corresponding antibody called isoagglutinin This is commonly used term in blood grouping 35 Explain how we used serological typing to identify S aureus Mix latex reagent by shaking expel any latex from the dropper for complete mixing Dispense 1 drop of Test Latex onto each circle of the reaction card Using a microbiological loop pick up and smear a colony to be tested on the test latex containing circle and mix it into the test latex reagent For the two remaining circles smear one colony of S aureus positive control and one colony of s epidermidis negative control Pick up and hand rock the card for up to 20 seconds and observe for agglutination under normal lighting conditions 36 Distinguish between a positive and negative test for an agglutination reaction Positive Results is obtained if agglutination of the blue latex occurs within 20 seconds in the test circle If agglutination occurs noticeable clearing of the blue background in the test latex will be observed This indicates the presence of S aureus Negative Results is obtained if no agglutination occurs and a smooth suspension remains at 20 seconds in the test circle If no agglutination occurs there will be no noticeable clearing of the blue background in the test latex
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